Eibai Lee

Anti-apoptotic Effect of cGMP in Cultured Astrocytes INHIBITION BY cGMP-DEPENDENT PROTEIN KINASE OF MITOCHONDRIAL PERMEABLE TRANSITION PORE

Reperfusion of cultured astrocytes with normal medium after exposure to H2O2-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H2O2-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H2O2-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca2+-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H2O2-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.

Apoptosis in Ca2 + reperfusion injury of cultured astrocytes: roles of reactive oxygen species and NF-κB activation

We previously reported that incubation of cultured astrocytes in Ca2 + ‐containing medium after exposure to Ca2 + ‐free medium caused Ca2 +  influx followed by delayed cell death. Here, we studied the mechanisms underlying the Ca2 + ‐mediated injury of cultured astrocytes. Our results show that Ca2 +  reperfusion injury of astrocytes appears to be mediated by apoptosis, as demonstrated by DNA fragmentation and prevention of death by caspase‐3 inhibitors. Paradoxical Ca2 +  challenge stimulated rapidly reactive oxygen species (ROS) production. Ca2 +  reperfusion injury of astrocytes was influenced by several reagents which modified ROS production. When astrocytes were exposed to hydrogen peroxide (H2O2) for 30 min and then incubated without H2O2 for 1–5 days, cell toxicity including apoptosis was observed. Ca2 +  reperfusion injury induced by Ca2 +  depletion or H2O2 exposure was blocked by the iron chelator 1,10‐phenanthroline, the NF‐κB inhibitor pyrrolidinedithiocarbamate and the calcineurin inhibitor FK506. Incubation in normal medium after H2O2 exposure rapidly increased the level of nuclear NF‐κB p65 subunit, and the effect was blocked by 1,10‐phenanthroline, pyrrolidinedithiocarbamate and FK506. These findings indicate that Ca2 +  reperfusion‐induced apoptosis is mediated at least partly by ROS production and ROS cause NF‐κB activation in cultured astrocytes.

Mechanism of inactivation of myeloperoxidase by propylthiouracil

The mechanism of inactivation of myeloperoxidase purified from rat bone marrow by propylthiouracil (PTU) was studied. PTU inhibited not only the peroxidase activity but also the chlorinating activity of myeloperoxidase in a concentration dependent manner. When myeloperoxidase was treated with PTU and hydrogen peroxide (5 microM), inactivation of the enzyme was still observed after the excess reagents were removed by a column of Sephadex G-25. The treatment of the enzyme with PTU in the absence of hydrogen peroxide caused a slight inhibition of the enzyme activity. In addition, [14C]PTU became bound to myeloperoxidase in the presence of hydrogen peroxide. Difference spectrum of myeloperoxidase incubated with the small (0.1 mM) and large (2 mM) amounts of hydrogen peroxide revealed the formation of compounds II and III, respectively. Difference spectrum of myeloperoxidase treated with PTU in the presence of a low concentration of hydrogen peroxide (5 microM) was similar to that of compound II. Therefore, these results indicate that PTU inactivates myeloperoxidase through binding to the enzyme and the conversion to a compound II-like form in the presence of hydrogen peroxide.

Ibudilast attenuates astrocyte apoptosis via cyclic GMP signalling pathway in an in vitro reperfusion model

We examined the effect of 3‐isobutyryl‐2‐isopropylpyrazolo[1,5‐a]pyridine (ibudilast), which has been clinically used for bronchial asthma and cerebrovascular disorders, on cell viability induced in a model of reperfusion injury. Ibudilast at 10 – 100 μM significantly attenuated the H2O2‐induced decrease in cell viability. Ibudilast inhibited the H2O2‐induced cytochrome c release, caspase‐3 activation, DNA ladder formation and nuclear condensation, suggesting its anti‐apoptotic effect. Phosphodiesterase inhibitors such as theophylline, pentoxyfylline, vinpocetine, dipyridamole and zaprinast, which increased the guanosine‐3′,5′‐cyclic monophosphate (cyclic GMP) level, and dibutyryl cyclic GMP attenuated the H2O2‐induced injury in astrocytes. Ibudilast increased the cyclic GMP level in astrocytes. The cyclic GMP‐dependent protein kinase inhibitor KT5823 blocked the protective effects of ibudilast and dipyridamole on the H2O2‐induced decrease in cell viability, while the cyclic AMP‐dependent protein kinase inhibitor KT5720, the cyclic AMP antagonist Rp‐cyclic AMPS, the mitogen‐activated protein/extracellular signal‐regulated kinase inhibitor PD98059 and the leukotriene D4 antagonist LY 171883 did not. KT5823 also blocked the effect of ibudilast on the H2O2‐induced cytochrome c release and caspase‐3‐like protease activation. These findings suggest that ibudilast prevents the H2O2‐induced delayed apoptosis of astrocytes via a cyclic GMP, but not cyclic AMP, signalling pathway.

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes.

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.

Inhibition of P-Glycoprotein by Wogonin Is Involved with the Potentiation of Etoposide-Induced Apoptosis in Cancer Cells

Etoposide induces apoptotic cell death in normal and cancer cells. This apoptosis plays a role not only in anticancer effects but also in adverse reactions, such as myelosuppression. Because we had previously found that wogonin, a flavone found in a plant, suppresses thymocyte apoptosis induced by etoposide, we examined the effect of this flavone in cancer cells. Wogonin significantly potentiated etoposide‐induced apoptosis in HL‐60 cells. This flavone impaired the function of P‐glycoprotein and then increased cellular content of etoposide in the cells. Thus, this flavone is likely to act as an inhibitor of P‐glycoprotein and potentiate the apoptotic action of etoposide. On the other hand, wogonin inhibited etoposide‐induced apoptosis in thymocytes, one of the normal cells. The potentiation by wogonin is likely to be a specific action for cancer cells but not normal cells. Therefore, this flavone may be used to reduce the excretion of the anticancer agents via P‐glycoprotein and increase the pharmacological action of it in cancer cells. These results suggest that wogonin may play a role in overcoming multidrug resistance.

Purification and some properties of peroxidases of rat bone marrow

Myeloperoxidase and eosinophil peroxidase were separated and purified from rat bone marrow cells using cetyltrimethylammonium bromide as the solubilizer and then with column chromatographies on CM-Sephadex C-50 and Con A-Sepharose. Both purified enzymes were observed to be apparently homogeneous by SDS-polyacrylamide gel electrophoresis. Myeloperoxidase consisted of two subunits of Mr 57,000 and 15,000, and eosinophil peroxidase two of 53,000 and 14,000. On structural analysis of the enzymes, their visual and ESR spectra revealed that the structure surrounding the heme in myeloperoxidase was different from that in eosinophil peroxidase. Moreover, substrate specificity and sensitivity to inhibitors such as azide and cyanide differed between the two enzymes. Rat bone marrow possesses two distinct peroxidases, myeloperoxidase and eosinophil peroxidase, which have different subunits and different heme microenvironments. Therefore, the difference in enzymatic function between the two peroxidases may be due to their structures.

Wogonin potentiates the antitumor action of etoposide and ameliorates its adverse effects

Wogonin, a flavone in the roots of Scutellaria baicalensis, reduced etoposide-induced apoptotic cell death in normal cells, such as bone marrow cells and thymocytes. On the other hand, wogonin potentiated the proapoptotic or cytotoxic action of etoposide in tumor cells, such as Jurkat, HL-60, A549, and NCI-H226. These contradictory actions of wogonin on apoptosis are distinguished by normal or cancer cell types. Wogonin had no effect on apoptosis induced by other anticancer agents in the tumor cells. Thus, the potentiation effect of wogonin was observed only in etoposide-induced apoptosis in tumor cells. In a functional assay for P-glycoprotein (P-gp), wogonin suppressed excretion of calcein, a substrate for P-gp, in these tumor cells. Moreover, wogonin decreased the excretion of radiolabeled etoposide and accordingly increased intracellular content of this agent in the cells. P-gp inhibitors showed a similar potentiation effect on etoposide-induced apoptosis in these tumor cells. Thus, wogonin is likely to potentiate the anticancer action of etoposide due to P-gp inhibition and accumulation of this agent. These findings suggest that wogonin may be a useful chemotherapeutic adjuvant to potentiate the pharmacological action of etoposide and ameliorate its adverse effects.

Oxidative metabolism of propylthiouracil by peroxidases from rat bone marrow.

1. Propylthiouracil (PTU) was degraded by myeloperoxidase (MPO) or eosinophil peroxidase (EPO), purified from rat bone marrow, in the presence of H2O2 and Cl-. In the absence of either H2O2 or Cl-, MPO and EPO do not degrade PTU. Optimum concentrations of KCl for MPO and EPO were 50 and 250 mM, respectively. 2. The characteristics of PTU degradation by MPO-H2O2-Cl- were similar to those of the chlorinating activity of the peroxidase. 3. Hypochlorous acid as well as MPO-H2O2-Cl- also degraded PTU. Metabolites of PTU degradation by MPO-H2O2-Cl-, which were separated by C18 reversed phase h.p.l.c., were the same as those produced by hypochlorous acid. 4. Of the metabolites of PTU formed by MPO-H2O2-Cl-, one was identified as PTU sulphonic acid (6-propyl-4-hydroxypyrimidine-2-sulphonate) and another seemed to be propyluracil.

Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution.

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.